Harnessing protein sensing ability of electrochemical biosensors via a controlled peptide receptor–electrode interface

Background Cathepsin B, a cysteine protease, is considered a potential biomarker for early diagnosis of cancer and inflammatory bowel diseases. Therefore, more feasible and effective diagnostic method may be beneficial for monitoring of cancer or related diseases. Results A phage-display library was biopanned against biotinylated cathepsin B to identify a high-affinity peptide with the sequence WDMWPSMDWKAE. The identified peptide-displaying phage clones and phage-free synthetic peptides were characterized using enzyme-linked immunosorbent assays (ELISAs) and electrochemical analyses (impedance spectroscopy, cyclic voltammetry, and square wave voltammetry). Feasibilities of phage-on-a-sensor, peptide-on-a-sensor, and peptide-on-a-AuNPs/MXene sensor were evaluated. The limit of detection and binding affinity values of the peptide-on-a-AuNPs/MXene sensor interface were two to four times lower than those of the two other sensors, indicating that the peptide-on-a-AuNPs/MXene sensor is more specific for cathepsin B (good recovery (86–102%) and %RSD (< 11%) with clinical samples, and can distinguish different stages of Crohn’s disease. Furthermore, the concentration of cathepsin B measured by our sensor showed a good correlation with those estimated by the commercially available ELISA kit. Conclusion In summary, screening and rational design of high-affinity peptides specific to cathepsin B for developing peptide-based electrochemical biosensors is reported for the first time. This study could promote the development of alternative antibody-free detection methods for clinical assays to test inflammatory bowel disease and other diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12951-023-01843-0.

EDC/NHS-functionalized gold electrode at 25 ℃ for 2 h and washed with distilled water to remove unbound or residual reagents and biotin-labeled synthetic peptides were added and incubated at 25 ℃ for 1 h. Then, the peptide-functionalized sensor was incubated in 1 % BSA for 15 min to decrease non-specific binding. The developed peptide on a sensor was reacted with cathepsin B and binding affinity was observed using SWV.

Generation of gold nanoparticle-embedded MXene (AuNPs-MXene)
Gold nanoparticles (AuNPs) were synthesized by chemical reduction of AuCl4by dissolving Na3Ctr at 100 ℃. First, 100 mL of 1.0 mM HAuCl4•3H2O containing 200 µL of 1 M NaOH was prepared in a 250 mL round flask. The solution was reacted at 100 ℃ under stirring, and 10 mL of 38.8 mM Na3Ctr was rapidly added in a reactor. The reaction was further proceeded for 15 min until changing color, and the synthesized AuNPs were cooled and stored at room temperature.
Ti3C2F MXene layers were synthesized via a conventional method. First, 1 g of LiF was dispersed in 7 M HCl solution and stirred continuously for 30 min. Then, 1.5 g of Ti3AlC2 MAX was slowly immersed in the solution and continuously stirred at 60 ℃ for 90 h under ambient conditions. The samples were then separated by centrifugation and redispersed in water to obtain a dispersed solution of neutral pH. The samples were finally collected through filtration, followed by drying for 24 h in a vacuum oven at 80 ℃.
AuNPs-MXene composites were synthesized by first dissolving 50 mg of Ti3C2F MXene layers in 35 mL of distilled water and were subjected to continuous ultrasonication for 3 h. Subsequently, 5 mL of AuNPs was additionally supplemented and ultrasonicated for extra collected by filtration, and the etched samples were dried for 24 h in a vacuum oven at 50 ℃.
The morphology and elemental distribution were observed using field-emission transmission electron microscopy (FE-TEM, JEOL, Japan) and high-resolution scanning electron microscopy (HR-SEM, HITACHI, Japan). The crystalline structure of fabricated AuNPs-MXene composites was determined by a multi-purpose X-ray diffractometer (MP-XRD; Pro MRD, RIGAKU, Japan), and the composition in AuNPs-MXene samples was analyzed by Fourier-transform infrared spectroscopy (FT-IR, JASCO, Japan) in a range with 400-4000 cm -1 . In order to confirm the chemical composition and the chemical states of nanocomposites, X-ray photoelectron spectroscopy measurements were performed.

Fabrication of peptide on a AuNPs-MXene sensor
The fabrication of AuNPs-MXene and immobilization of peptides on the electrode was done by the following steps. Initially, AuNPs-MXene paste was prepared by sonication AuNPs-MXene suspension (4 mg/mL) containing 0.1 % nafion solution for 1 h [3,4]. Prior to modification, the gold electrode was polished with piranha solution (H2O2:H2SO4 = 1:3) and washed with distilled water. After that, 15 μL of AuNPs-MXene with 0.1 % nafion was dropped on the polished gold electrode surface and incubated at 25 ℃ for 3 h. After complete drying, the electrode was immersed in 1 mM MUA overnight and washed with distilled water. The MUA-immobilized electrode was functionalized with 400 mM/100 mM of EDC/NHS and then 100 μg/mL streptavidin solution was added and incubated for 2 h. Then, synthetic peptides were added to functionalized electrode and incubated for 1 h. The peptidefunctionalized AuNPs-MXene sensor was incubated in 1 % BSA for 15 min to decrease nonspecific binding. Finally, peptide on the AuNPs-MXene sensor was interacted with cathepsin B and its binding affinity was investigated using SWV.    Designed to see the effect of negative charged residue on binding interaction Incorporated biotin at C-termini for specific immobilization via streptavidin-biotin interaction